In vitro metabolic stability assays using rainbow trout (Oncorhynchus mykiss) isolated hepatocytes (RT-HEP) or hepatic S9 fractions (RT-S9) were introduced to provide biotransformation rate data for the assessment of chemical bioaccumulation in fish. The present study explored the suitability of the RT-HEP and RT-S9 assays for difficult test chemicals, and the in vitro-based predictions were compared to in silico-based predictions and in vivo-measured bioconcentration factors (BCFs). The results show that volatile or reactive chemicals can be tested with minor modifications of the in vitro protocols. For hydrophobic chemicals, a passive dosing technique was developed. Finally, a design-of-experiment approach was used to identify optimal in vitro assay conditions. The modified assay protocols were applied to 10 fragrances with diverse physicochemical properties. The in vitro intrinsic clearance rates were higher in the S9 than in the hepatocyte assay, but the in vitro-in vivo (IVIV) predictions were comparable between the 2 assays. The IVIV predictions classified the test chemicals as nonbioaccumulative (BCF < 2000), which was in agreement with the in vivo data but in contrast to the in silico-based predictions. The findings from the present study provide strong evidence that the RT-HEP and RT-S9 assays can provide reliable estimates of in vivo biotransformation rates for test chemicals with difficult physicochemical properties. Environ Toxicol Chem 2020;39:2396-2408. © 2020 SETAC.